Tuberculosis, an infectious disease caused by the microorganism Mycobacterium tuberculosis, continues to remain a major global health problem. In the United States, tuberculosis still persists as a significant health problem, particularly in underprivileged and minority populations, among immigrants from high-risk countries, and in other high-risk groups, such as individuals with human immunodeficiency viral infections.
A definitive diagnosis of tuberculosis depends upon the isolation of Mycobacterium tuberculosis (sometimes referred to hereinafter as "M. tuberculosis") from the secretions or tissues of an infected individual in conjunction with clinical findings of the disease. Because of the length of time required for isolation of M. tuberculosis and the subsequent diagnosis of tuberculosis, a long-standing goal of researchers has been to develop a rapid, sensitive, and specific test for the detection of the organism in clinical specimens. Such a test could substantially decrease the time required to definitively diagnose tuberculosis, and assist the health care provider in administering the appropriate therapeutic treatment.
A variety of methods have been used for detection of M. tuberculosis. With culture identification procedures, a specimen is typically placed on an acceptable culture medium, incubated at a specific temperature for a period of time, and then inspected for growth of the organism. Although routinely performed, these procedures are highly technical, expensive, and laborious.
A second method, direct microscopy, involves the direct examination of smears prepared from a clinical specimen. Although the most rapid method for detecting mycobacteria, direct microscopy is limiting because technical expertise is required in interpreting smears and a large number of bacteria must be present for detection.
Non-cultural methods, such as radioimmunoassay, latex agglutination and enzyme immunosorbent assay, have also been employed for the direct detection of M. tuberculosis. These approaches appear to be promising because of their rapidity. However, a major limitation with these methods is the lack of sensitivity in detecting the organism.
A more promising diagnostic approach has been achieved with recombinant DNA and hybridization techniques. Nucleic acid hybridization assays have been used to detect and identify target genetic materials such as DNA in clinical specimens. See Tenover, Clin. Microbiol. Rev. 1:82-101, 1988. The assay is premised upon the presence of a specific nucleotide sequence in the target genetic material, and the detection of this sequence. The direct detection of the specific nucleotide sequence actually occurs by use of a nucleic acid probe. A probe is a nucleotide sequence that is complementary to the DNA sequence of one of the strands of the DNA molecule that is desired to be detected in the clinical sample. A detectable marker is attached to the probe.
DNA probe technology offers advantages over other detection methods. The detection time is shorter, and the technology is not dependent upon the viability of the organism. Despite these benefits, a significant limitation of this technology is its lack of sensitivity in detecting M. tuberculosis.
A DNA probe system is available for detecting species of mycobacteria, but only after the organism has grown in culture (Gen-Probe, Inc.). Gonzalez et al., Diagn. Microbiol. Infect. Dis., 8:69-77, 1987. However, no direct probe for the detection of mycobacteria in clinical specimens is currently available.